The Fbw7 protein functions as the substrate-recognition component of SCF ubiquitin ligases. Fbw7 promotes the degradation of several proteins implicated in hematopoietic stem cell (HSC) biology, including Myc, Notch, cyclin E, and c-Jun. Our preliminary data indicate that Fbw7 is expressed in HSCs/progenitors and regulates hematopoietic differentiation. The overall goal of this project is to understand the role of the Fbw7 pathway in hematopoietic stem/progenitor cell proliferation and differentiation, and to determine the importance of specific Fbw7 substrates in these processes. The Fbw7 gene encodes three protein isoforms that function in different subcellular compartments. The goal of Aim One is to examine the expression and regulation of the Fbw7 isoforms in HSCs/progenitors, mature hematopoietic elements, and during hematopoietic differentiation. The goal of Aim Two is study the functions of Fbw7 in HSCs/progenitors by using two complementary strategies to inactivate Fbw7 expression. The first approach utilizes shRNA-mediated Fbw7 knockdown to reduce Fbw7 expression in HSCs/progenitors in vitro, whereas the second approach entails the development of a conditional-null Fbw7 knockout mouse that will be used to inactivate Fbw7 in HSCs/progenitors in vivo. We will use these two strategies to study Fbw7 functions in hematopoietic stem/progenitor cells, and to determine the role of the Fbw7 pathway in regulating specific substrates such as Myc, Notch, and cyclin E in these cells. The goal of Aim Three is to understand the mechanisms that regulate Fbw7-mediated Notch degradation. We will initially use a biochemical approach to identify the phosphorylation events that signal Notch degradation by Fbw7. We will than study how this pathway is regulated in HSCs/progenitors by developing antibodies that recognize these key phosphorylation(s). Finally, we will examine the importance of Fbw7-mediated Notch degradation in HSCs/progenitors by developing a conditional "knockin" mouse model in which Notch cannot be degraded by Fbw7 because it cannot be phosphorylated. Importantly, this model will specially impair Notch degradation while leaving the rest of the Fbw7 pathway intact. Overall this research will provide a comprehensive investigation of Fbw7 function in HSCs/progenitors.